User:Karmella Haynes/Notebook/BioBrick cloning/2011/05/21

From OpenWetWare

Jump to: navigation, search
Project name Main project page
Previous entry      Next entry

05/21/11

  • ✓ Minipreps: KAH181, KAH57 (r.t.)
  • ✓ Assembly: KAH182



Minipreps
> Check with E/P digests

Reagent Volume Expected:
1,2. KAH181 = 1444
3,4. KAH57 (r.t.) = 1426
Miniprep digests 5/21/11
15 μL/lane; 1% agarose -->
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.


--> Combine samples 3/4


Assemblies

  1. KAH182: KAH181/(S/P)/~4644 + polyA2/(X/P)/225

> Digests (Fermentas FD)

Reagent Volume   Cloning digest 5/21/11
30 μL/lane, 1% agarose
DNA (plasmid) up to 25 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH181 (X/P) 0.229 1.47 11.4


> Ligations

Ligation Plate results (lig : neg crtl) 05/22/11
1. polyA2(X/P)/225, 2 ng + KAH181(S/P)/~4644, 25 ng KAH182 >10:1 (Pick 4)
2. KAH181(S/P)/ 25 ng  
  1 2
Insert DNA 1.0 ---
Vector DNA 2.0 2.0
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O 1.0 2.0
  10 μL 10 μL


---> Add 10 μL to 30 μL DH5α



Personal tools